The present invention provides a nucleotide sequence encoding carbamoyl phosphate synthetase II of Plasmodium falciparum. carbamoyl phosphate synthetase II catalyses the first committed and rate-limiting step in the de novo pyrimidine biosynthetic pathway. P. falciparum relies exclusively on pyrimidine synthesis de novo because of its inability to salvage pyrimidines. Mature human red blood cells, however, have no recognized requirement for a pyrimidine nucleotide. Accordingly, this enzyme represents a prime chemotherapeutic locus. The present invention relates to the use of the sequence encoding carbamoyl phosphate synthetase II in the recombinant production of carbamoyl phosphate synthetase II and to antisense molecules, ribozymes and other gene inactivation agents designed from this sequence.
|
3. An antisense oligonucleotide capable of blocking expression of the nucleic acid molecule as shown in SEQ ID No:1.
1. A ribozyme capable of cleaving carbamoyl phosphate synthetase II mRNA, the ribozyme including sequences complementary to portions of mRNA obtained from the nucleic acid molecule as shown in SEQ ID NO:1.
2. The ribozyme as claimed in
4. A polynucleotide construct which produces in a cell the ribozyme as claimed in
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This application is a continuation-in-part of application Ser. No. 08/446,885, filed Jul. 6, 1995, now U.S. Pat. No. 5,849,573.
The present invention relates to nucleotide sequences encoding carbamoyl phosphate synthetase II of Plasmodium falciparum, to methods of producing this enzyme using recombinant DNA technology and to the use of this sequence and enzyme in the design of therapeutics.
The urgency for the design of novel chemotherapeutic agents for the treatment of malaria has been renewed in recent times due to the evolution of human malarial parasites, primarily Plasmodium falciparum, which are resistant to traditional drugs. Research into a vaccine seems a very plausible alternative, but after years of investigation, no clinically acceptable product has come to date. At the same time, there is also an increasing decline in the efficacy of insecticides against mosquito vectors. At present, more than two-thirds of the world's population--approximately 500 million people--are thought to live in malaria areas (Miller, 1989). It ranks eighth in the World Health Organization's (WHO) list of ten most prevalent diseases of the world (270 million infections a year) and ranks ninth of the ten most deadly diseases, claiming over 2 million lives a year (Cox, 1991; Marshall, 1991). Though chiefly confined to poor nations, there are recent reports of infections in the United States (Marshall, 1991) and Australia (Johnson, 1991), and ever increasing cases of travellers' malaria (Steffen and Behrens, 1992).
Comparative biochemical studies between the malaria parasite, P. falciparum and its host have revealed differences in a number of metabolic pathways. One such distinction is that the parasite relies exclusively on pyrimidine synthesis de novo because of its inability to salvage preformed pyrimidines (Sherman, 1979). Moreover, the mature human red blood cell has no recognised requirement for pyrimidine nucleotides (Gero and O'Sullivan, 1990). Major efforts have been directed towards the development of inhibitors of the pyrimidine biosynthetic pathway (Hammond et al., 1985; Scott et al., 1986; Prapunwattana et al., 1988; Queen et al., 1990; Krungkrai et al., 1992), confirming its potential as a chemotherapeutic locus. Current research into the molecular biology of the key pyrimidine enzymes is envisioned as a powerful tool, not only to get a better understanding of the parasite's biochemistry, but also to explore specific differences between the parasite and the mammalian enzymes.
Glutamine-dependent carbamoyl phosphate synthetase (CPSU, EC 6.3.5.5) catalyses the first committed and rate-limiting step in the de novo pyrimidine biosynthetic pathway of eukaryotic organisms (Jones, 1980). Moreover, because it catalyzes a complex reaction involving three catalytic units and several substrates and intermediates, it is a very interesting enzyme to study from a biochemical point of view. The structural relationship of CPSII to other pyrimidine enzymes varies in different organisms, making it a good subject for evolutionary studies.
The paucity of material that can be obtained from malarial cultures has hampered the isolation of adequate amounts of pure protein for analysis. The difficulty in purifying CPS is further augmented by its inherent instability. Studies using crude extracts from P. berghei (a rodent malaria) revealed a high molecular weight protein containing CPS activity, which was assumed to be associated with ATCase (Hill et al., 1981), a situation also found in yeast (Makoff and Radford, 1978). However, recent analysis by Krungkrai and co-workers (1990) detected separate CPSII and ATCase activities in P. berghei. Although CPS activity has been detected in P. falciparum (Reyes et al., 1982) until this current study there is no indication of its size nor its linkage with other enzymes in the pathway.
The glutamine-dependent activity of CPSII can be divided into two steps: (1) a glutaminase (GLNase) reaction which hydrolyzes glutamine (Gln) and transfers ammonia to the site of the carbamoyl phosphate synthetase; and (2) a synthetase reaction. where carbamoyl phosphate is synthesised from two molecules of adenosine triphosphate (ATP), bicarbonate and ammonia. The second activity involves three partial reactions: (a) the activation of bicarbonate by ATP; (b) the reaction of the activated species carboxyphosphate with ammonia to form carbamate; and (c) the ATP-dependent phosphorylation of carbamate to form carbamoyl phosphate (powers and Meister, 1978). Hence, there are two major domains in CPSII, the glutamine amidotransferase domain (GAT) and the carbamoyl phosphate synthetase domain (CPS) or simply synthetase domain. The glutaminase domain (GLNase) is a subdomain of GAT, while there are two ATP-binding subdomains in the synthetase domain.
In view of the similarities between the glutamine amidotransferase domain of CPS and other amidotransferases, it has been proposed that these subunits arose by divergent evolution from a common ancestral gene (=20 kDa) representing the GLNase domain and that particular evolution of the CPS GAT domain (=42 kDa which includes the putative structural domain only present in CPS) must have involved fusions and/or insertions of other sequences (Werner et al., 1935). The GAT of mammalian CPSI gene has been proposed to be formed by a simple gene fusion event at the 5' end of this ancestral gene with an unknown gene (Nyunoya et al., 1985).
The genes for the larger synthetase domains of various organisms were postulated to have undergone a gene duplication of an ancestral kinase gene resulting in a polypeptide with two homologous halves (Simmer et al., 1990). Unlike the subunit structure of E. coli and arginine-specific CPS of yeast, a further fusion of the genes encoding GAT and the synthetase domains was suggested to have formed the single gene specific for pyrimidine biosynthesis in higher eukaryotes. Conversely, Simmer and colleagues (1990) proposed that the arginine-specific CPS's (like cpa1 and cpa2 in yeast) as well as rat mitochondrial CPSI arose by defusion from the pyrimidine chimera.
The present inventors have isolated and characterised the complete gene encoding the CPSII enzyme from P. falciparum (pfCPSII). Reported here is the sequence including 5' and 3' untranslated regions. In so doing, the present inventors have identified the respective glutaminase and synthetase domains. Unlike CPSII genes in yeast, D. discoideum, and mammals, there is no evidence for linkage to the subsequent enzyme, aspartate transcarbamoylase (ATCase). This is in contrast to the report by Hill et al., (1981) for the enzymes from P. berghei. The present inventors have, however, found two large inserts in the P. falciparum gene of a nature that does not appear to have been previously described.
Accordingly, in a first aspect, the present invention consists in a nucleic acid molecule encoding carbamoyl phosphate synthetase II of Plasmodium falciparum, the nucleic acid molecule including a sequence substantially as shown in Table 1 from 1 to 7176, or from 1 to 750, or from 751 to 1446, or from 1447 to 2070, or from 2071 to 3762, or from 3763 to 5571, or from 5572 to 7173, of from 1 to 3360, or from 2071 to 6666, or from 2071 to 7173, or a functionally equivalent sequence.
In a preferred embodiment of the present invention, the nucleic acid molecule includes a sequence shown in Table 1 from -1225 to 7695 or a functionally equivalent sequence.
In a second aspect, the present invention consists in an isolated polypeptide, the polypeptide including an amino acid sequence substantially as shown in Table 1 from 1 to 2391, from 483 to 690, from 691 to 1254, 1858 to 2391, from 1 to 1120, from 691 to 2222, or from 691 to 2391.
As used herein the term "functionally equivalent sequence" is intended to cover minor variations in the nucleic acid sequence which, due to degeneracy in the code, do not result in the sequence encoding a different polypeptide.
In a third aspect the present invention consists in a method of producing Plasmodium falciparum carbamoyl phosphate synthetase II, the method comprising culturing a cell transformed with the nucleic acid molecule of the first aspect of the present invention under conditions which allow expression of the nucleic acid sequence, and recovering the expressed carbamoyl phosphate synthetase II.
The cells may be either bacteria or eukaryotic cells. Examples of preferred cells include E.coli, yeast, and Dictyostelium discoideum.
As will be readily understood by persons skilled in this field, the elucidation of the nucleotide sequence for CPSII enables the production of a range of therapeutic agents. These include antisense nucleotides, ribozymes, and the targeting of RNA and DNA sequences using other approaches, e.g., triplex formation.
As can be seen from a consideration of the sequence set out in Table 1 the Plasmodium falciparum CPSII gene includes two inserted sequences not found in other carbamoyl phosphate synthetase genes. The first inserted sequence separates the putative structural domain and the glutiminase domain whilst the second inserted sequence separates the two ATP binding subdomains of the synthetase subunit CPSa and CPSb.
| TABLE 1 |
| Nucleotide and Deduced |
| Amino Acid Sequence of the Carbamoyl Phosphate |
| Synthetase II Gene from Plasmodium falciparum [SEQ ID NOS:1 and 2] |
| . . . . . . |
| -1225 GAATTCCTTCAGCCAAAAAAAATGACAACGCAAATTTTAAGAAAAGAAAAACAATCGACT |
| -1156 |
| . . . . . . |
| -1165 CGTCTTTGAATGAGGTTAGAAATTCGATACGTGAAAGGGACTTAAGAAGGCTTAACAGAG |
| -1106 |
| . . . . . . |
| -1105 AAAAGAGTAAAATCTTATAAGCATTTGAAGGAAAAAATAATAAAATAAAAAAATAAAAAG |
| -1046 |
| . . . . . . |
| -1045 ATAAAAAATATTTATATTTGATATGTAGTATATATAATGATTATTCATATTAATAACATA |
| -986 |
| . . . . . . |
| -985 GATAAAAAACTTTTTTTTTTTTTTTTTTTCTTTATATTTATTAACAATACATTTAAGTTA |
| -926 |
| . . . . . . |
| -925 TTTTATATATATATATATATATATATATATATATATATATATATATGTTTGTGTGTTCAT |
| -866 |
| . . . . . . |
| -865 TTGTTTATAAAATTACTTGAAATATAAAACTTATTAATATATTTCCAATTAATATGAATA |
| -806 |
| . . . . . . |
| -805 CAATTATTAATATTTTCATGTGTACACATTAATATAGTTTTACACTTCTTATAATAAAAC |
| -746 |
| . . . . . . |
| -745 CATCCTATATATTATACACAATATATAATACTCCCCAATATTGTGGTTCCTATAATTTTA |
| -686 |
| . . . . . . |
| -685 TTTATATATTTATTTATTAATTTATTCATTTATTTATTTTTTTTCTTAGTTTATAAAATA |
| -626 |
| . . . . . . |
| -625 GTAATTCTACTAATTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAATT |
| -566 |
| . . . . . . |
| -565 TACATATGAAAAATGAACTTGTATATGTAAATTTATAAATATTTTAAACATAAATATAAA |
| -506 |
| . . . . . . |
| -505 TGTATAAAAAAAAAAAAGAAAAATGGGAAAAAATAATATAGATATATATATAAATATATA |
| -446 |
| . . . . . . |
| -445 TATATATATAATTATTGGGGATATTCTCTGAATCATAGGTCTTAAACAGTTTTATTCTTT |
| -385 |
| . . . . . . |
| -385 TAACATCACAAAGTTGTTATTAAAAGTATATATATCTTATTGGTTCCTATATAAAACTAT |
| -326 |
| . . . . . . |
| -325 AGTATTCTATAATATATTCTGTATATTTCATTTTATCATTTGTAAGCAATCCCTATTTAT |
| -266 |
| . . . . . . |
| -265 TATAATTATTATTTTTTTTTTTATAAAAGAGGTATAAAACAGTTTATTCAATTTTTTTCC |
| -206 |
| . . . . . . |
| -205 TAAAGGAGCAACCTTCAGTCAATTTACATTTTCCACCGGTTGGTTGGCACAACATAATGT |
| -146 |
| . . . . . . |
| -145 TACAGCTAAAAAAAGAAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATATATATAT -86 |
| . . . . . . |
| -85 ATATATATATATATACATAATATGTACAATGCTACCATACAAGTATATAAATTTTTCAAC -26 |
| . . |
| -25 ATTGTTGTGATGTTGCATTTTTCTT -1 |
| . . . . . . |
| 1 ATGTATATTTCTTTTAAATATAATTTATATATATATATATATATATATATATATATATTT 60 |
| 1 M Y I S F K Y N L Y I Y I Y I Y I Y I F 20 |
| . . . . . . |
| 61 GTTCTTATAGATTTTAAAACAGTTGGGAGGTTAATTCTTGAAGATGGTAACGAATTTGTA 120 |
| 21 V L I D F K T V G R L I L E D G N E F V 40 |
| . . . . . . |
| 121 GGGTACAGTGTAGGTTACGAAGGGTCTAAAGGAAATAATAGTATATCATGTCATAAGGAG 180 |
| 41 G Y S V G Y E G C K G N N S I S C H K E 60 |
| . . . . . . |
| 181 TATAGAAATATTATTAATAATGATAATAGCAAGAATAGTAATAATTCATTTTGTAATAAT 240 |
| 61 Y R N I I N N D N S K N S N N S F C N N 80 |
| . . . . . . |
| 241 GAACAAAACAATTTGAAAGATCATTTATTATATAAAAATAGTCGATTAGAAAATGAAGAT 300 |
| 81 E E N N L K D D L L Y K N S R L E N E D 100 |
| . . . . . . |
| 301 TTTATTGTTACAGGTGAAGTTATATTTAATACAGCTATGGTTGGATATCCTGAAGCTTTA 360 |
| 101 F X V T G E V Z F N T A M V G Y P E A L 120 |
| . . . . . . |
| 361 ACGGACCCAAGTTATTTTGGTCAAATATTAGTTTTAACATTTCCTTCTATTGGTAATTAT 420 |
| 121 T D P S Y F G Q I L V L T F P S I G N Y 140 |
| . . . . . . |
| 421 GGTATTGAAAAAGTAAAACATGATGAAACGTTTGGATTAGTACAAAATTTTGAAAGTAAT 480 |
| 141 G I E K V K H D E T F G L V Q N F E S N 160 |
| . . . . . . |
| 481 AAAATTCAAGTACAAGGTTTAGTTATTTGTGAATATTCGAAGCAATCATATCATTACAAT 540 |
| 161 K X Q V Q G L V X C E Y S K Q S Y H Y N 180 |
| . . . . . . |
| 541 TCTTATATTACCTTAAGTGAATGGTTAAAGATTTATAAAATTCCATGTATAGGTGCTATA 600 |
| 181 S Y I T L S E W L K I Y K I P C I G G I 200 |
| . . . . . . |
| 601 GATACAAGAGCCTTAACAAAACTTTTAAGAGAAAAAGGTAGTATGTTAGGTAAAATACTT 660 |
| 201 D T R A L T K L L R E K G S M L G K I V 220 |
| . . . . . . |
| 661 ATATATAAAAACAGACAACATATTAATAAATTATATAAAGAAATTAATCTTTTTGATCCT 720 |
| 221 I Y K N R Q H I N K L Y K E I N L F D P 240 |
| . . . . . . |
| 721 GGTAATATAGATACTCTAAAATATGTATGTAATCATTTTATACGTGTTATTAAGTTGAAT 780 |
| 241 G N I D T L K Y V C N H F I R V I K L N 260 |
| . . . . . . |
| 781 AATATTACATATAATTATAAAAATAAGGAAGAATTTAATTATACCAATGAAATGATTACT 840 |
| 261 N I T Y N Y K N K E E F N Y T N E N I T 280 |
| . . . . . . |
| 841 AATGATTCTTCAATGGAAGATCATGATAATGAAATTAATGGTAGTATTTCTAATTTTAAT 900 |
| 281 N D S S M E D H D N Z I N G S I S N F N 300 |
| . . . . . . |
| 901 AATTGTCCAAGTATCTCTAGTTTTGATAAAAGTGAATCGAAAAATGTTATTAATCATACA 960 |
| 301 N C P S I S S F D K S E S K N V I N H T 320 |
| . . . . . . |
| 961 TTGTTAAGAGATAAAATGAACCTAATAACTTCATCTGAAGAATATCTGAAACATCTTCAT |
| 1020 |
| 321 L L R D K M N L I T S S E E Y L K D L H 340 |
| . . . . . . |
| 1021 AATTGTAATTTTAGTAATAGTAGTGATAAAAATGATTCTTTTTTTAAGTTATATGGTATA |
| 1080 |
| 341 N C N F S N S S D K N D S F F K L Y G I 360 |
| . . . . . . |
| 1021 TGTGAATATGATAAATATTTAATTGACCTTGAAGAAAATGCTAGCTTTCATTATAATAAT |
| 1140 |
| 351 C E Y D K Y L I D L E E N A S F H Y N N 380 |
| . . . . . . |
| 1141 GTAGATGAATATGGATATTATGATGTTAATAAAAATACAAATATTCTATCTAATAATAAA |
| 1200 |
| 381 V D E Y G Y Y D V N K N T N I L S N N K 400 |
| . . . . . . |
| 1201 ATAGAACAAAACAACAATAACGAAAATAACAAAAATAACAAAAATAACAACAATAACGAG |
| 1260 |
| 401 I E Q N N N N Z N N K N N K N N N N N E 420 |
| . . . . . . |
| 1261 GTTGATTATATAAAGAAAGATGAGGATAATAATGTCAATAGTAAGGTCTTTTATAGCCAA |
| 1320 |
| 421 V D Y I K K D E D N N V N S K V F Y S Q 440 |
| . . . . . . |
| 1321 TATAATAATAATGCACAAAATAATGAACATACCGAATTTAATTTAAATAATGATTATTCT |
| 1380 |
| 441 Y N N N A Q N N E H T E F N L N N D Y S 460 |
| . . . . . . |
| 1381 ACTTATATTAGAAAGAAAATGAAAAATCAAGAATTCCTTAATTTGGTAAACAAAAGAAAA |
| 1440 |
| 461 T Y I R K K M K N E E F L N L V N K R K 480 |
| . . . . . . |
| 1441 GTAGACCATAAAGAAAAAATTATTGTTATTGTTGATTGTGGTATTAAAAATAGTATAATC |
| 1500 |
| 481 V D H K E K I I V I V D C G I K N S I I 500 |
| . . . . . . |
| 1501 AAAAATTTAATAAGACACGGTATGGATCTTCCATTAACATATATTATTGTACCTTATTAT |
| 1560 |
| 501 K N L I R H G M D L P L T Y I I V P Y Y 520 |
| . . . . . . |
| 1561 TACAATTTTAATCATATAGATTATGATGCAGTTCTTTTATCTAATGGTCCTGGAGATCCT |
| 1620 |
| 521 Y N F N H I D Y D A V L L S N G P G D P 540 |
| . . . . . . |
| 1621 AAAAAGTGTGATTTCCTTATAAAAAATTTGAAAGATAGTTTAACAAAAAATAAAATTATA |
| 1680 |
| 541 K K C D F L I K N L K D S L T K N K I I 560 |
| . . . . . . |
| 1681 TTTGGTATTTGTTTAGGTAATCAACTATTAGGTATATCATTAGGTTGTGACACATATAAA |
| 1740 |
| 561 F G I C L G N Q L L G I S L G C D T Y K 580 |
| . . . . . . |
| 1741 ATGAAATATGGTAATAGAGGTGTTAATCAACCCGTAATACAATTAGTAGATAATATATGT |
| 1800 |
| 581 M K Y G N R G V N Q P V I Q L V D N I C 600 |
| . . . . . . |
| 1801 TACATTACCTCACAAAATCATGGATACTGTTTAAAGAAAAAATCAATTTTAAAAAGAAAA |
| 1860 |
| 601 Y I T S Q N H G Y C L K K K S I L K R K 620 |
| . . . . . . |
| 1861 GAGCTTGCGATTAGTTATATAAATGCTAATGATAAATCTATACAAGGTATTTCACATAAA |
| 1920 |
| 621 E L A I S Y I N A N D I S I E G I S H K 640 |
| . . . . . . |
| 1921 AATGGAAGATTTTATAGTGTCCAGTTTCATCCTCAGGGTAATAATGGTCCTGAAGATACA |
| 1980 |
| 641 N G R F Y S V Q F H F E G N N G P E D T 660 |
| . . . . . . |
| 1981 TCATTTTTATTTAACAATTTTCTTTTAGATATCTTTAATAAGAAAAAACAATATAGAGAA |
| 2040 |
| 661 S F L F K N F L L D I F N K K K Q Y R E 680 |
| . . . . . . |
| 2041 TATTTAGGATATAATATTATTTATATAAAAAAGAAAGTGCTTCTTTTAGGTAGTGGTGGT |
| 2100 |
| 681 Y L G Y N I I Y I K K K V L L L G S G G 700 |
| . . . . . . |
| 2101 TTATGTATAGGACAAGCAGCACAATTCGATTATTCAGGAACA(AAGCAATTAAAAGTTTA |
| 2160 |
| 701 L C I G Q A G E F D Y S G T Q A I K S L 720 |
| . . . . . . |
| 2161 AAAGAATGTGGTATATATGTTATATTAGTTAATCCTAACATAGCAACTGTTCAAACATCA |
| 2220 |
| 721 K E C G I Y V I L V N F N I A T V Q T S 740 |
| . . . . . . |
| 2221 AAAGGTTTGGCAGATAAGGTATACTTTTTACCAGTTAATTGTGAATTTGTAGAAAAAATT |
| 2280 |
| 741 K G L A D K V Y F L P V N C E F V E K I 760 |
| . . . . . . |
| 2281 ATTAAAAAGGAAAAACCTGATTTTATTTTATGTACATTTGGTGGTCACACAGCTTTAAAT |
| 2340 |
| 761 I K K E K P D F I L C T F G G Q T A L N 780 |
| 2341 TGTGCTTTAATGTTAGATCAAAAAAAAGTATTGAAAAAGAATAATTGTCAATGTTTAGGT |
| 2400 |
| 781 C A L M L D Q K K V L K K N N C C C L G 800 |
| . . . . . . |
| 2401 ACATCTTTAGAATCTATAAGAATAACAGAAAATAGAACATTATTTGCTGAAAAATTAAAA |
| 2460 |
| 801 T S L E S I R I T E N R T L F A E K L K 820 |
| . . . . . . |
| 2461 GAAATTAATGAAAGAATAGCTCCATATGGTAGTGCAAAAAATGTTAATCAAGCTATTGAT |
| 2520 |
| 821 E I N E R I A P Y G S A K N V N Q A I D 840 |
| . . . . . . |
| 2521 ATAGCTAATAAAATAGGATATCCAATATTAGTACGTACAACATTTTCGTTAGGAGGATTA |
| 2580 |
| 841 I A N K I G Y P I L V R T T F S L G G L 860 |
| . . . . . . |
| 2581 AATAGTAGTTTCATAAATAATGAAGAAGAACTTATCGAAAAATGTAATAAAATATTTTTA |
| 2640 |
| 861 N S S F I N N E E E L I E K C N K I F L 880 |
| . . . . . . |
| 2641 CAAACTGATAATGAAATATTTATAGATAAATCATTACAAGGATGGAAAGAAATAGAATAT |
| 2700 |
| 881 Q T D N E T F I D K S L Q G W K E I E Y 900 |
| . . . . . . |
| 2701 GAATTATTAAGAGATAATAAAAATAATTGTATAGCTATATGTAATATGGAAAATATAGAT |
| 2760 |
| 901 E J L R D N K N N C I A I C N M E N I D 920 |
| . . . . . . |
| 2761 CCATTAGGTATACATACAGGAGATAGTATAGTTGTTGCACCTTCACAAACATTAAGTAAT |
| 2820 |
| 921 P L G I H T G D S I V V A P S Q T L S N 940 |
| . . . . . . |
| 2821 TATGAATATTATAAATTTAGAGAAATAGCATTAAAGGTAATTACACATTTAAATATTATA |
| 2880 |
| 941 Y L Y Y K F R E I A L K V I T H L N I I 960 |
| . . . . . . |
| 2881 GGAGAATGTAATATACAATTTGGTATAAATCCACAAACAGGAGAATATTGTATTATTGAA |
| 2940 |
| 961 G E C N I Q F G I N P Q T G E Y C I I E 980 |
| . . . . . . |
| 2941 GTTAATGCTAGGCTTAGTAGAAGTTCAGCATTACCTTCTAAAGCTACTGGTTATCCACTT |
| 3000 |
| 981 V N A R L S R S S A L A S K A T G Y P L 1000 |
| . . . . . . |
| 3001 GCTTATATATCAGCAAAAATAGCCTTGGGATATGATTTGATAAGTTTAAAAAATAGCATA |
| 3060 |
| 1001 A Y I S A K I A L G Y D L I S L K N S I 1020 |
| . . . . . . |
| 3061 ACTAAAAAAACAACTGCCTGTTTTGAACCCTCTCTAGATTACATTACAACAAAAATACCA |
| 3120 |
| 1021 T K K T T A C F E P S L D Y I T T K I P 1040 |
| . . . . . . |
| 3121 CGATCGGATTTAAATAAATTTGAGTTTGCTTCTAATACAATGAATAGTAGTATGAAAAGT |
| 3180 |
| 1041 R W D L N K F E F A S N T M N S S M K S 1060 |
| . . . . . . |
| 3181 GTAGGAGAAGTTATGTCTATAGGTAGAACCTTTGAAGAATCTATACAAAAATCTTTAAGA |
| 3240 |
| 1061 V G E V M S I G R T F E E S I Q K S L R 1080 |
| . . . . . . |
| 3241 TGTATTGATGATAATTATTTAGGATTTAGTAATACGTATTGTATAGATTGGGATGAAAAG |
| 3300 |
| 1081 C I D D N Y L G F S N T Y C I D W D E K 1100 |
| . . . . . . |
| 3301 AAAATTATTCAAGAATTAAAAAATCCATCACCAAAAAGAATTGATGCTATACATCAAGCT |
| 3360 |
| 1101 K I I E E L K N P S P K R I D A I H Q A 1120 |
| . . . . . . |
| 3361 TTCCATTTAAATATGCCTATGGATAAAATACATGAGCTGACACATATTGATTATTGGTTC |
| 3420 |
| 1121 F H L N M P M D K I H E L T H I D Y W F 1140 |
| . . . . . . |
| 3421 TTACATAAATTTTATAATATATATAATTTAGAAAATAAGTTGAAAACGTTAAAATTAGAG |
| 3480 |
| 1141 L H K F Y N I Y N L Q N K L K T L K L E 1160 |
| . . . . . . |
| 3481 CAATTATCTTTTAATGATTTGAAGTATTTTAAGAAGCATGGTTTTAGTGATAAGCAAATA |
| 3540 |
| 1161 Q L S F N D L X Y F K K W G F S D K Q I 1180 |
| . . . . . . |
| 3541 GCTCACTACTTATCCTTCAACACAAGCGATAATAATAATAATAATAATAATATTAGCTCA |
| 3600 |
| 1181 A H Y L S F N T S D N N N N N N N I S S 1200 |
| . . . . . . |
| 3601 TGTAGGGTTACAGAAAATGATGTTATGAAATATA&AGAAAAGCTAGGATTATTTCCACAT 3660 |
| 1201 C R V T E N D V M K Y R E K L G L F P H 1220 |
| . . . . . . |
| 3661 ATTAAAGTTATTGATACCTTATCAGCCGAATTTCCGGCTTTAACTAATTATTTATATTTA |
| 3720 |
| 1221 I K V I D T L S A E F P A L T N Y L Y L 1240 |
| . . . . . . |
| 3721 ACTTATCAAGGTCAAGAACATGATGTTCTCCCATTAAATATGAAAAGGAAAAAGATATGC |
| 3780 |
| 1241 T Y Q G Q E H D V L P L N M K R K K I C 1260 |
| . . . . . . |
| 3781 ACGCTTAATAATAAACGAAATGCAAATAAGAAAAAAGTCCATGTCAAGAACCACTTATAT |
| 3840 |
| 1261 T L N N K R N A N K K K V R V K N X L Y 1280 |
| . . . . . . |
| 3841 AATGAAGTAGTTGATGATAAGGATACACAATTACACAAAGAAAATAATAATAATAATAAT |
| 3900 |
| 1281 N E V V D D K D T Q L H K E N N N N N N 1300 |
| . . . . . . |
| 3901 ATGAATTCTGGAAATGTAGAAAATAAATGTAAATTGAATAAAGAATCCTATGGCTATAAT |
| 3960 |
| 1301 M N S G N V E N K C K L N K E S Y G Y N 1320 |
| . . . . . . |
| 3961 AATTCTTCTAATTGTATCAATACAAATAATATTAATATAGAAAATAATATTTGTCATGAT |
| 4020 |
| 1321 N S S N G I N T N N I N I E N N I C H D 1340 |
| . . . . . . |
| 4021 ATATCTATAAACAAAAATATAAAAGTTACAATAAACAPLTTCCAATAATTCTATATCAAT |
| 4080 |
| 1341 I S I N K N I K V T I N N S N N S I S N 1360 |
| . . . . . . |
| 4081 AATGAAAATGTTGAAACAAACTTAAATTGTGTATCTGAAAGGGCCGGTAGCCATCATATA |
| 4140 |
| 1361 N E N V E T N L N C V S E R A G S H H I 1380 |
| . . . . . . |
| 4141 TATGGTAAAGAAGAAAAGAGTATAGGATCTGATGATACAAATATTTTAAGTGCACAAAAT |
| 4200 |
| 1381 Y G K E E K S I G S D D T N I L S A C N 1400 |
| . . . . . . |
| 4201 TCAAATAATAACTTTTCATGTAATAATGAGAATATGAATAAAGCAAACGTTGATGTTAAT |
| 4260 |
| 1401 S N N N F S C N N E N M N K A N V D V N 1420 |
| . . . . . . |
| 4261 GTACTAGAAAATGATACGAAAAAACGAGAAGATATAAATACTACAACAGTATTTATGGAA |
| 4320 |
| 1421 V L E N D T K K R E D I N T T T V F N E 1440 |
| . . . . . . |
| 4321 GGTCAAAATAGTGTTATTAATAATAAGAATAAAGAGAATAGTTCTTTATTGAAAGGTGAT |
| 4380 |
| 1441 G Q N S V I N N K N K E N S S L L K G D 1460 |
| . . . . . . |
| 4381 GAAGAAGATATTGTGATGGTAAATTTAAAAAAGGAAAATAATTATAATAGTGTAATTAAT |
| 4440 |
| 1461 E E D I V N V N L K K E N N Y N S V Y N 1480 |
| . . . . . . |
| 4441 AATGTAGATTGTAGGAAAAAGGATATGGATGGAAAAAATATAAATGATGAATGTAAAACA |
| 4500 |
| 1481 N V D C R K K D M D G K N I N D E C K T 1500 |
| . . . . . . |
| 4501 TATAAGAAAAATAAATATAAAGATATGGGATTAAATAATAATATAGTAGATGAGTTATCC |
| 4560 |
| 1501 Y K K N K Y K D M G L N N N I V D E L S 1520 |
| . . . . . . |
| 4561 AATGGAACATCACATTCAACTAATGATCATTTATATTTAGATAATTTTAATACATCAGAT |
| 4620 |
| 1521 N G T S H S T N D H L Y L D N F N T S D 1540 |
| . . . . . . |
| 4621 GAAGAAATAGGGAATAATAAAAATATGGATATGTATTTATCTAAGGAAAAAAGTATATCT |
| 4680 |
| 1541 E E I G N N K N M D M Y L S K E K S I S 1560 |
| . . . . . . |
| 4681 AATAAAAACCCTGGTAATTCTTATTATGTTGTAGATTCCGTATATAATAATGAATACAAA |
| 4740 |
| 1561 N K N P G N S Y Y V V D S V Y N N E Y K 1580 |
| . . . . . . |
| 4741 ATTAATAAGATGAAAGAGTTAATAGATAACGAAAATTTAAATGATGAATATAATAATAAT |
| 4800 |
| 1581 I N K M K E L I D N E N L N D E Y N N N 1600 |
| . . . . . . |
| 4801 GTTAATATGAATTGTTCTAATTATAATAATGCTAGTGCATTTGTAAATGGAAAGGATAGA |
| 4860 |
| 1601 V N M N C S N Y N N A S A F V N G K D K 1620 |
| . . . . . . |
| 4861 AATGATAATTTAGAAAATGATTGTATTGAAAAAAATATGGATCATACATACAAACATTAT |
| 4920 |
| 1621 N D N L E N D C I E K N M D H T Y K H Y 1640 |
| . . . . . . |
| 4921 AATCGTTTAAACAATCGTAGAAGTACAAATGAGAGGATGATGCTTATGGTAAACAATGAA |
| 4980 |
| 1641 N R L N N R R S T N E R M M L M V N N E 1660 |
| . . . . . . |
| 4981 AAAGAGAGCAATCATGAGAAGGGCCATAGAAGAAATGGTTTAAATAAAAAAAATAAAGAA |
| 5040 |
| 1661 K E S N H E K C H R R N G L N K K N K E 1680 |
| . . . . . . |
| 5041 AAAAATATGGAAAAAAATAAGGGAAAAAATAAAGACAAAAAGAATTATCATTATGTTAAT |
| 5100 |
| 1681 K N M E K N K G K N K D K K N Y H Y V N 1700 |
| . . . . . . |
| 5101 CATAAAAGGAATAATGAATATAATAGTAACAATATTGAATCGAAGTTTAATAATTATGTT |
| 5160 |
| 1701 H K R N N E Y N S N N I E S K F N N Y V 1720 |
| . . . . . . |
| 5161 GATGATATAAATAAAAAAGAATATTATGAAGATGAAAATGATATATATTATTTTACACAT |
| 5220 |
| 1721 D D I N K K E Y Y E D E N D I Y Y F T H 1740 |
| . . . . . . |
| 5221 TCGTCACAAGGTAACAATGACGATTTAAGTAATGATAATTATTTAAGTAGTGAAGAATTG |
| 5280 |
| 1741 S S Q G N N D D L S N D N Y L S S E E L 1760 |
| . . . . . . |
| 5281 AATACTGATGAGTATGATGATGATTATTATTATGATGAACATGAAGAAGATGACTATGAC |
| 5340 |
| 1761 N T D E Y D D D Y Y Y D E D E E D D Y D 1780 |
| . . . . . . |
| 5341 GATGATAATGATGATGATGATGATGATGATGATGATGGGGAGGATGAGGAGGATAATGAT |
| 5400 |
| 1781 D D N D D D D D D D D D G E D E E D N D 1800 |
| . . . . . . |
| 5401 TATTATAATGATGATGGTTATGATAGCTATAATTCTTTATCATCTTCAAGAATATCAGAT |
| 5460 |
| 1801 Y Y N D D G Y D S Y N S L S S S R I S D 1820 |
| . . . . . . |
| 5461 GTATCATCTGTTATATATTCAGGGAACGAAAATATATTTAATGAAAAATATAATGATATA |
| 5520 |
| 1821 V S S V I Y S G N E N T F N E K Y N D I 1840 |
| . . . . . . |
| 5521 GGTTTTAAAATAATCGATAATAGGAATGAAAAAGAGAAAGAGAAAAAGAAATGTTTTATT |
| 5580 |
| 1841 G F K I I D N R N E K E K E K K K C F I 1860 |
| . . . . . . |
| 5581 GTATTAGGTTGTGGTTGTTATCGTATTGGTAGTTCTGTAGAATTTGATTGGAGTGCTATA |
| 5640 |
| 1861 V L G C G C Y R I Q S S V E F D W S A I 1880 |
| . . . . . . |
| 5641 CATTGTGTAAAGACCATAAGAAAATTAAACCATAAAGCTATATTAATAAATTGTAACCCA |
| 5700 |
| 1881 H C V K T I R K L N H K A I L T N C N F 1900 |
| . . . . . . |
| 5701 GAAACTGTAAGTACAGATTATGATGAAAGTGATCGTCTATATTTTGATGAAATAACAACA |
| 5760 |
| 1901 E T V S T D Y D E S D R L Y F D E I T T 1920 |
| . . . . . . |
| 5761 GAAGTTATAAAATTTATATATAACTTTGAAAATAGTAATGGTGTGATTATAGCTTTTGGT |
| 5820 |
| 1921 E V I K F I Y N F E N S N G V I I A F G 1940 |
| . . . . . . |
| 5821 GGACAAACATCAAATAATTTAGTATTTAGTTTATATAAAAATAATGTAAATATATTACGA |
| 5880 |
| 1941 G Q T S N N L V F S L Y K N N V N I L G 1960 |
| . . . . . . |
| 5881 TCAGTGCACAAAGTGTTGATTCTTGTGAAAATAGGAATAAATTTTCGCACTTATGTGATT |
| 5940 |
| 1961 S V H K V L I V V K I G I N F R T Y V I 1980 |
| . . . . . . |
| 5941 CTTAAAATTGATCAACCGAAATGGAATAAATTTACAAAATTATCCAAGGCTATACAATTT |
| 6000 |
| 1981 L K I D Q P K W N K F T K L S K A I Q F 2000 |
| . . . . . . |
| 6001 GCTAATGAGGTAAAATTTCCTGTATTAGTAAGACCATCGTATGTATTATCTGGTGCAGCT |
| 6060 |
| 2001 A N E V K F F V L V R P S Y V L S G A A 2020 |
| . . . . . . |
| 6061 ATGAGAGTTGTAAATTGTTTTGAAGAATTAAAAAACTTTTTAATGAAGGCAGCTATTGTT |
| 6120 |
| 2021 M R V V N C F E E L K N F L M K A A I V 2040 |
| . . . . . . |
| 6121 AGTAAAGATAATCCTGTTCTAATATCAAAATTTATTGAGAATGCTAAAGAAATAGAAATA |
| 6180 |
| 2041 S K D N P V V I S K F T E N A K E I E I 2060 |
| . . . . . . |
| 6181 GATTGTGTTAGTAAAAATGGTAAAATAATTAATTATGCTATATCTGAACATGTTGAAAAT |
| 6240 |
| 2061 D C V S K N G K I I N Y A I S E H V E N 2080 |
| . . . . . . |
| 6241 CCTGGTGTACATTCAGGTGATCCAACATTAATATTACCTGCACAAAATATATATGTTGAA |
| 6300 |
| 2081 A G V H S C D A T L I L P A Q N I Y V E 2100 |
| . . . . . . |
| 6301 ACACATAGGAAAATAAAGAAAATATCCGAAAACATTTCAAAATCATTAAATATATCTGGT |
| 6360 |
| 2101 T H R K I K K I S E K I S K S L N I S G 2120 |
| . . . . . . |
| 6361 CCATTTAATATACAATTTATATCTCATCAAAATGAAATAAAAATTATTGAATGTAATTTA |
| 6420 |
| 2121 P F N I Q F I C H Q N E I K I I E C N L 2140 |
| . . . . . . |
| 6421 AGAGCATCTAGAACTTTTCCATTTATATCAAAAGCTCTAAATCTAAACTTTATAGATTTA |
| 6480 |
| 2141 R A S R T F P F I S K A L N L N F I D L 2160 |
| . . . . . . |
| 6481 GCTACAAGGATATTAATGGGTTATGACGTCAAACCAATTAATATATCATTAATTGATTTA |
| 6540 |
| 2161 A T R I L N G Y D V K P I N I S L I D L 2180 |
| . . . . . . |
| 6541 GAATATACAGCTGTAAAAGCACCGATTTTCTCATTTAATAGATTACATGGATCAGATTGT |
| 6600 |
| 2181 E Y T A V K A P I F S F N R L H G S D C 2200 |
| . . . . . . |
| 6601 ATACTAGGTGTAGAAATGAAATCTACAGGTGAAGTAGCATGTTTTGGTTTAAATAAATAT |
| 6660 |
| 2201 I L C V E M K S T G E V A C F G L N K Y 2220 |
| . . . . . . |
| 6661 GAAGCTTTATTAAAATCATTAATAGCTACAGGTATGAAGTTACCCAAAAAATCAATACTT |
| 6720 |
| 2221 E A L L K S L I A T G M K L F K K S I L 2240 |
| . . . . . . |
| 6721 ATAAGTATTAAAAATTTAAATAATAAATTAGCTTTTGAAGAACCGTTCCAATTATTATTT |
| 6780 |
| 2241 I S I K N L N N K L A F E E P F C L L F 2260 |
| . . . . . . |
| 6781 TTAATGGGATTTACAATATATGCGACTCAAGGTACGTATCATTTCTACTCTAAATTTTTA |
| 6840 |
| 2261 L N G F T I Y A T E G T Y D F Y S K F L 2280 |
| . . . . . . |
| 6841 CAATCTTTTAATCTTAATAAAGGTTCTAAATTTCATCAAACACTTATTAAAGTTCATAAT |
| 6900 |
| 2281 E S F N V N K G S K F H Q R L I K V H N 2300 |
| . . . . . . |
| 6901 AAAAATGCAGAAAATATATCACCAAATACAACAGATTTAATTATGAATCATAAAGTTGAA |
| 6960 |
| 2301 K N A E N I S P N T T D L I M N H K V E 2320 |
| . . . . . . |
| 6961 ATGGTTATTAATATAACTGATACATTAAAAACAAAGGTTAGTTCAAATGGTTATAAAATT |
| 7020 |
| 2321 M V I N I T D T L K T K V S S N G Y K I 2340 |
| . . . . . . |
| 7021 AGAAGATTAGCATCAGATTTCCAGGTTCCTTTAATAACTAATATGAAACTTTGTTCTCTT |
| 7080 |
| 2341 R R L A S D F Q V P L I T N M K L C S L 2360 |
| . . . . . . |
| 7081 TTTATTGACTCATTATATAGAAAATTCTCAAGACAAAAGGAAAGAAAATCATTCTATACC |
| 7140 |
| 2361 F I D S L Y R K F S R Q K E R K S F Y T 2380 |
| . . . |
| 7141 ATAAAGAGTTATGACGAATATATAAGTTTGGTATAA |
| 7176 |
| 2381 I K S Y D E Y I S L V * 2392 |
| . . . . . . |
| 7177 GCAAGAAATTATTCAATAAATTCGATTTAACATTACTTATTTATGTATTTATTAACTTTC |
| 7235 |
| . . . . . . |
| 7237 ATTCCATAACAACATGAAAAGTATAAATATATAAATAGTAATATATAATATATAATATAT |
| 7296 |
| . . . . . . |
| 7297 ATATATATATATATATATATATTTATTTATTTAATTATATTTACGTTTAAATATTAATAA |
| 7356 |
| . . . . . . |
| 7357 ATGTTTTTATTAAATATGATCATTAATTTATATTGATTTATTTTTTTATAAATTTTTGTT |
| 7416 |
| . . . . . . |
| 7417 ATATATACAAATTTTATTTATTCACTCATATGTATAAACCAAAATGGTTTTTTCAATTTA |
| 7476 |
| . . . . . . |
| 7477 CAAATAATTTTATAATTTTAATAAATTTATTAATTATAAAAAAAATAAAAATATATAAAC |
| 7536 |
| . . . . . . |
| 7537 ATTAAAATGTATAAATTCTTTTAATTATATAATAATTTATAAATGTTATGATTTTTTTAA |
| 7596 |
| . . . . . . |
| 7597 AAAATTCAACGAAAAAAAAGAGGAACTGTATATACAAAAGGGACTATATATATGTATATA |
| 7656 |
| . . . |
| 7657 TATATATATATATATATGTTTTTTTTTCCTTATTCTAGA |
| 7695 |
The GAT domain is made up of two subdomains: a putative structural domain (1-750) and a glutaminase domain (1447-2070). These two subdomains are separated by a first inserted sequence (751-1446, underlined). The two ATP binding subdomains of the synthetase subunit, CPSa (2071-3762) and CPSb (5572-5173) are separated by a second inserted sequence (3763-5571, underlined).
As these inserted sequences are not found in other carbamoyl phosphate synthetase genes they represent prime targets for therapies including, but not limited to, antisense nucleotides, ribozymes and triplex forming nucleotides as there is a decreased likelihood of deleterious reaction with host homologues of the gene.
Antisense RNA molecules are known to be useful for regulating gene expression within the cell. Antisense RNA molecules which are complementary to portion(s) of CPSII can be produced from the CPSII sequence. These antisense molecules can be used as either diagnostic probes to determine whether or not the CPSII gene is present in a cell or can be used as a therapeutic to regulate expression of the CPSII gene, Antisense nucleotides prepared using the CPSII sequence include nucleotides having complementarity to the CPSII mRNA and capable of interfering with its function in vivo and genes containing CPSII sequence elements that can be just transcribed in living cells to produce antisense nucleotides. The genes may include promoter elements from messenger RNA (polymerase II) from cells, viruses, pathogens or structural RNA genes (polymerase I & III) or synthetic promoter elements. A review of antisense design is provided in "Gene Regulation; Biology of Antisense RNA and DNA" R. P. Erickson and J. G. Izant, Raven Press 1992. Reference may also be had to U.S. Pat. No. 5,208,149 which includes further examples on the design of antisense nucleotides. The disclosure of each of these references is incorporated herein by reference.
As used herein the term "nucleotides" include but are not limited to oligomers of all naturally-occurring deoxyribonucleotides and ribonucleotides as well as any nucleotide analogues. Nucleotide analogues encompass all compounds capable of forming sequence-specific complexes (eg duplexes or hetroduplexes) with another nucleotide including methylphosphonates or phosphorothioates but may have advantageous diffusion or stability properties. The definition of nucleotides includes natural or analogue bases linked by phosphodiester bonds, peptide bonds or any other covalent linkage. These nucleotides may be synthesised by any combination of in vivo in living cells, enzymatically in vitro or chemically.
Ribozymes useful in regulating expression of the CPSII gene include nucleotides having CPSII sequence for specificity and catalytic domains to promote the cleavage of CPSII mRNA in vitro or in vivo. The catalytic domains include hammerheads, hairpins, delta-virus elements, ribosome RNA introns and their derivatives. Further information regarding the design of ribozymes can be found in Haseloff, J. & Gerlach, W. L. (1988) Nature 334. 585; Kruger, K., Grabowski, P. J., Zaug, A. J., Sands, J., Gottschling, D. E. & Cech, T. R. (1982) Cell 31, 147; International Patent Application No. WO 88/04300, U.S. Pat. No. 4,987,071 and U.S. Pat. No. 5,254,678. The disclosure of each of these references is incorporated herein by reference. The catalytic elements may enhance the artificial regulation of a CPSII target mRNA by accelerating degradation or some other mechanism.
Triple helix oligonucleotides can be used to inhibit transcription from the genome. Given the sequence provided herein for the CPSII gene it will now be possible to design oligonucleotides which will form triplexes thereby inhibiting transcription of the CPSII gene. Information regarding the generation of oligonucleotides suitable for triplex formation can be found in a Griffin et al (Science 245:967-971 (1989)) this disclosure of this reference is incorporated herein by reference.
Triplex agents include all nucleotides capable of binding to the CPSII gene through formation of the complex with DNA or chromatin. The interaction can be through formation of a triple-stranded Hoogsteen structure or other mechanisms such as strand invasion that relies on the CPSII sequence information.
Accordingly, in a fourth aspect the present invention consists in a ribozyme capable of cleaving carbamoyl phosphate synthetase II mRNA, the ribozyme including sequences complementary to portions of mRNA obtained from the nucleic acid molecule of the first aspect of the present invention.
In a preferred embodiment of this aspect of the present invention the ribozyme includes sequences complementary to mRNA obtained from the first or second inserted sequences of the nucleic acid molecule of the first aspect of the present invention.
In a fifth aspect the present invention consists in an antisense oligonucleotide capable of blocking expression of the nucleic acid molecule of the first aspect of the present invention.
As stated above, in one aspect the present invention relates to a method of producing CPSII by recombinant technology. The protein produced by this method and the polypeptides of the present invention will be useful in in vitro drug binding studies in efforts to develop other anti-malarial therapeutics.
In order that the nature of the present invention may be more clearly understood the method by which the P. falciparum CPSII gene was cloned will now be described with reference to the following Examples and Figures.
FIG. 1: A summary of a "gene walking" strategy used to clone and sequence the full length P. falciparum carbamoyl phosphate synthetase II gene.
FIG. 2: P. falciparum carbamoyl phosphate synthetase II (pfCPSII) gene sequence with the 21 consensus GUC (GTC) ribozyme cleavage sites identified (underlined) (SEQ ID NO:1).
FIG. 3: Output of RNA mfold analysis showing the GUC sites from CPSRz1/M10 a and CPSRz4/M15 more accessible than the M17 and M18 sites (Seq. Id. No:1).
FIGS. 4A-C: A. Sequences for the phosphorothioated antisense DNA used in inhibition studies of P. Falciparum in culture (SEQ ID NOS:3-15); B. Map of the positions of the antisense sequences within the pfCPSII gene; C. Growth supression of P. Falciparum in vitro after a 24 hr incubation with the oligonucleotides.
FIG. 5. Cultures of P. Falciparum and selected mammalian cell lines were incubated with 2.5 and 5.0 μM CPSRz1, CPSRz4 and 60-mer random oligonucleotide. Cell viability was assessed by measuring the % incorporation of tritiated hypoxanthine.
Cloning of the P. falciparum Carbamoyl Phosphate Synthetase II (pfCPSII) Gene
The conventional way to screen for genes of which the amino acid sequence had not been previously determined is via heterologous probing, i.e. with gene fragments of the target enzyme from closely related organisms. This has proved to be unsuccessful for several workers with Plasmodium falciparum largely due to the unusually high A-T content of its genome. After initial unfruitful attempts to isolate the CPSII gene in Plasmodium falciparum using a yeast ura2 gene fragment (Souciet et al., 1989), the present inventors opted to amplify part of the CPSII gene using the polymerase chain reaction (PCR) (Saiki et al., 1988) with a view to use the amplified product as probe for screening.
The present inventors isolated and cloned a PCR product using oligonucleotides designed from conserved sequences from the amino terminal GAT domain and the first half of the synthetase domain of the CPS gene. Nucleotide sequencing confirmed that a portion of the CPSII gene had been obtained. Total parasite DNA was fragmented with a restriction enzyme and subjected to Southern analysis using the CPSII-specific gene probe. The sizes of DNA fragments hybridizing to the gene probe were determined then the DNA in the corresponding bands were used for the construction of a "mini-library". In this way a smaller population of clones were screened for the pf CPSII gene.
To isolate the full length pfCPSII gene, a series of mini-libraries were constructed utilising different segments of known sequence to gain information of the unknown flanking regions both towards the 5' and 3' termini of the gene using "gene-walking". The strategy employed is summarised in FIG. 1.
In the first Southern analysis, total P. falciparum DNA was digested with HindIII and EcoRI and hybridisation was carried out using the pfCPSII 453 bp PCR product. A 3.0 kb HindIII and a smaller EcoRI fragment hybridised to the probe. Subsequent screening of a HindIII pTZ18U mini-library resulted in the isolation of a recombinant that contained a 3.0 kb pfCPSII gene fragment, CPS2. The 453 bp PCR product was localised in the middle of this segment.
Two regions from both the 5' and 3' ends of CPS2 were used to isolate neighbouring sequences at either end in order to obtain the further gene sequences. A HindIII/EcoRI fragment from the 5' end of CPS2 was instrumental in isolating a further 1.5 kb fragment, CPS1 consisting of the complete 5' region of the gene and some non-encoding sequences.
A 550 bp inverse PCR (IPCR; Triglia et al., 1988) product was obtained with the aid of known sequences from the 3' end of CPS2.
This IPCR product was used to screen for the 3' region flanking CPS2, A 3.3 kb HindIII recombinant containing CPS3 as well as a related 3.3 kb XBaI clone (not presented in FIG. 1) were isolated by the mini-library technique. Using a 200 bp XbaI/HindIII fragment from the 3' end of CPS 3, a 1.3 kb XbaI segment, CPS4 was cloned which contained the putative stop codon and some 3' non-coding region.
Combining these four gene fragments (CPS1, CPS2, CPS3 and CPS4) excluding their overlaps, gives a total of 8.8 kb consisting of approximately 7.0 kb coding and 1.8 flanking sequences.
The complete nucleotide sequence of the CPSII gene in P. falciparum, together with its 5' and 3' flanking sequences, is presented in Table 1.
Design of Ribozymes
Our first generation ribozymes were designed using the consensus sequences cleavage site, GUC, found in naturally occurring hammerhead ribozymes (see Haseloff and Gerlach, 1988, the entire contents of which are incorporated herein by reference). In total, there are 21 GUC sites in the entire 7.1 kb coding region of pfCPSII (FIG. 1 and Table 2). To select the putative sites that are relatively more accessible to binding, the pfCPSII mRNA folding pattern was analysed using mfold Program (ANGIS/GCG). This was done in windows of 500 nucleotides, with 250 nucleotide overlaps between each window.
To screen for the best (most accessible) sites within the CPS II mRNA, a series of antisense DNA oligonucleotides were designed and synthesised from sequences around the selected (by mfold) GUC sequences mentioned above. Also included were two antisense oligonucleotides (M17 and M18) where the mfold program had indicated intramolecular base pairing of the GUC sequences may be occurring (FIG. 2). As seen from the bio-assays of suppression of malarial growth by the antisense oligonucleotides, there appears to be a correlation between the predicted accessibility of each site and the effectiveness as judged by the % inhibition of growth (FIG. 3). The antisense oligonucleotides M10 (Rz1 site) and M15 (Rz4 site) were much more effective in growth inhibition than M17 and M18 where the mfold program predicted internal base pairing.
These results led to the synthesis of ribozymes based on the Rz1 and Rz4 sites (ribozymes CPSRz1 and CPSRz4).
| TABLE 2 |
| GUC sites present in the coding region of pfCPSII. Nucleotide |
| numbers indicate the position of the G of the triplet in the gene sequence. |
| 1. 170 |
| 2. 281 |
| 3. 380 |
| 4. 905 CPSRz1 site/M10 |
| 5. 1294 |
| 6. 1306 |
| 7. 1607 |
| 8. 1939 |
| 9. 1967 |
| 10. 2324 |
| 11. 2387 |
| 12. 3195 |
| 13. 3731 CPSRz4 site/M15 |
| 14. 3817 |
| 15. 3823 |
| 16. 4013 |
| 17. 432Z |
| 18. 5223 M17 |
| 19. 5735 |
| 20. 6359 |
| 21. 6383 |
CPSRz1 and CPSRz4 as Antimalarials
The ability of CPSRz1 and CPSRz4 to cleave a 550 base mRNA fragment of pfCPSII was assessed. Growth inhibition studies on P. falciparum cultures were initially conducted using higher concentrations of ribozymes, at 2.5 and 5.0 μM. Both ribozymes were shown to be very effective at these concentrations as shown in the marked decrease in incorporation of tritiated hypoxanthine (FIG. 4). In the same study, a series of mammalian cell lines in culture were treated with the same concentrations of the ribozymes, and no effect cell viability was observed. As a negative control, a 60-mer DNA oligonucleotide of random sequence was used.
As will be readily appreciated by those skilled in the art the isolation of this gene and its sequencing by the present inventors opens up a range of new avenues for treatment of Plasmodium falciparum infection. The present invention enables the production of quantities of the Plasmodium falciparum carbamoyl phosphate synthetase II enzyme using recombinant DNA technology. Characterisation of this enzyme may enable its use as a chemotherapeutic loci.
The isolation of this gene also will enable the production of antisense molecules, ribozymes or other gene inactivation agents which can be used to prevent the multiplication of the parasite in infected individuals.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
Cox, F. E. G. (1991) Malaria vaccines: while we are waiting. Parasitology Today 7: 189-190
Gero, A. M. and O'Sullivan, W. J. (199)) Purines and pyrimidines in malarial parasites. Blood Cells 16: 467-498
Hammond, D. J. Burchell, J. R. and Pudney, M. (1965) Inhibition of pyrimidine biosynthesis de novo in Plasmodium falciparum by 2.(4.t-butylcyclohexyl)-3-hydroxy-1, 4-naphthoquinine in vitro. Mol. Biochem. Parasitol 14: 97-109
Haseloff, J. And Gerlach, W. L. (1988) Simple RNA enzymes with new and highly specific endoribonuclease activity. Nature 334:585-591
Hill, B., Kilsby, J. Rogerson, G. W., McIntosh, R T. and Ginger, C. D. (1981). The Enzymes of pyrimidine biosynthesis in a range of parasitic protozoa and helminths. Mol. Biochem. Parasitol. Z: 123-134.
Johnson, C. Malaria back to plague us. Sydney Morning Herald, Nov. 13, 1991.
Jones, M. E. (1980) Pyrimidine nucleotide biosynthesis in animals: genes, enzymes and regulation of UMP biosynthesis. Annu. Rev. Biochem. 49: 253-279.
Krungkrai, J. Cerami, A. and Henderson, G. B. (1990) Pyrimidine biosynthesis in parasitic protozoa: purification of a monofunctional dihydroorotase from Plasmodium berghei and Crithidia fasciculata. Biochemistry 29: 6270-6275.
Krungkrai, J. Krungkrai, S. R. and Phakanont, K. (1992) Antimalarial activity of orotate analogs that inhibit dihydrootase and dihydroorotate dehydrogenase. Biochem. Pharmacol. 43: 1295-1301.
Marshal, E. (1991) Malaria parasite gaining ground against science. Science 2: 190,
Nyunoya, H., Broglie, K. E., Widgren, W. E. and Lusty C. J (1985) Characterization and derivation of the gene coding for mitochondrial carbamyl phosphate synthetase I of rat. J. Biol. Chem. 260: 9346-9356.
Prapunwattana, P., O'Sullivan, W. J. and Yuthavong, Y. (1988) Depression of Plasmodium falciparum dihydroorotate dehydrogenase activity in in vitro culture by tetracycline. Mol. Biochemi. 27: 119-124.
Queen, S. A., Vander Jagt, D. L. and Reyes, P. (1990) In vitro susceptibilities of Plasmodium falciparum to compounds which inhibit nucleotide metabolism. Antimicrob. Agents Chemother. 34: 1393-1398.
Reyes, P., Rathod, P. K., Sanchez, D. J. Mrema, J. E. K., Rieckmann, K. H. and Heidrich, H. G. (1982) Enzymes of purine and pyrimidine metabolism from the human malaria parasite, Plasmodium falciparum. Mol. Biochem. Parasitol. 5: 275-290.
Rubino S. D., Nyunoya, H. and Lusty, C. J. (1986) JBC 261(24):11320-11327.
Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis K. B. and Erlich H. A. (1958) Science 239:487-491,
Scott, H. V., Gero, A. M. and O'Sullivan, W. J. (1986) In vitro inhibition of Plasmodium falciparum by pyrazofurin, an inhibitor of pyrimidine biosynthesis de novo. Mol Biochem. Parasitol. 18: 3-15.
Sherman, I. W. (1979) Biochemistry of Plasmodium (malarial parasites) Microbiol. Rev. 43: 453-495.
Simmer, J. P., Kelly, R. E., Rinker, Jr., A. G., Scully, J. L. and Evans D. R. (1990) Mammalian carbamyl phosphate synthetase (CPS), J. Biol. Chem 265: 10395-10402.
Simmer, J. P., Kelly, R. E., Austin, G. R., Jr., Scully, J. L. and Evans, D. R. (1990) JBC 285(18):10395-10402.
Souciet, J. L., Nagy, M., Le Gouar, M., Lacroute, F. and Potier, S. (1989) Gene (Amst.) 79: 59-70.
Triglia, T., Peterson, M. G. and Kemp, D. J. (1988) PNAS 16:8186.
Werner, M., Feller, A. and Pierard, A. (1985) Nucleotide sequence of yeast genie CPA1 encoding the small subunit of arginine-pathway carbamoyl-phosphate synthetase. Eur. J. Biochem. 146: 371-381.
Stewart, Thomas S., Flores, Maria V., O'Sullivan, William J.
| Patent | Priority | Assignee | Title |
| 11046945, | Feb 27 2013 | Cornell University | Labeled glutaminase proteins, isolated glutaminase protein mutants, methods of use, and kit |
| Patent | Priority | Assignee | Title |
| 5585479, | Jul 24 1992 | United States of America as represented by the Secretary of the Navy | Antisense oligonucleotides directed against human ELAM-I RNA |
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